Effect of oxygen supply on milk-clotting activity expressed by Paenibacillus spp. strain BD3526

•Syntheses of milk-clotting enzyme and exopolysaccharides by BD3526 are aerobic.•Oxygen supply favors the syntheses of milk-clotting enzyme and exopolysaccharides.•Exopolysaccharides accumulations hinder the elevation of milk-clotting activity.•BD3526 milk-clotting enzyme is a potential bacterial coagulant.

Paenibacillus spp. BD3526, a novel bacteria species isolated from raw yak (Bos grunniens) milk collected in Tibet, is able to produce metalloproteinase with milk-clotting activity (MCA) in wheat bran broth. To determine the critical factor favoring enzyme productivity, response surface methodology and the Box-Behnken design were adopted to preliminarily optimize BD3526 cultivation conditions for maximum MCA in shaking flasks. The highest degree of MCA (7072 ± 519 SU/mL) was obtained at a liquid volume of 30 mL and 300 r/min, which implies that oxygen supply is crucial for enzyme synthesis. MCA levels were enhanced as agitation speeds and aeration rates were increased in a 7-L scale fermentor. MCA in the supernatant was further boosted by adding hydrogen peroxide (H2O2) to the broth during cultivation. Maximum MCA (14778 ± 1100 SU/mL) and MCA/proteolytic activity (PA) (33000 ± 918) were achieved when cultivation was carried out at an agitation speed of 400 r/min, aeration rate of 5.5 L/min and H2O2 flow of 500 mmol/L at a feeding rate of 2.5 mL/h after 24 h of cultivation.